BECN1 F121A mutation increases autophagic flux in aged mice and improves aging phenotypes in an organ-dependent manner

Autophagy is necessary for lifespan extension in multiple model organisms and autophagy dysfunction impacts age-related phenotypes and diseases. Introduction of an F121A mutation into the essential autophagy protein BECN1 constitutively increases basal autophagy in young mice and reduces cardiac and renal age-related changes in longer-lived Becn1F121A mutant mice. However, both autophagic and lysosomal activity have been described to decline with age. Thus, whether autophagic flux is maintained during aging and whether it is enhanced in Becn1F121A mice is unknown. Here we demonstrate that old wild type mice maintained functional autophagic flux in heart, kidney and skeletal muscle but not liver, and old Becn1F121A mice had increased autophagic flux in those same organs compared to wild type. In parallel, Becn1F121A mice were not protected against age-associated hepatic phenotypes but demonstrated reduced skeletal muscle fiber atrophy. These findings identify an organ-specific role for the ability of autophagy to impact organ aging phenotypes.

Diagnostic Approaches to Various Muscle Diseases Based on Muscle Pathology

Muscle pathology can give much information to reach the diagnosis of neuromuscular disorders. Major pathological changes occurred in skeletal muscles include muscle fiber atrophy/hypertrophy, necrosis/regeneration, inflammation, myofibrillar disorganization, abnormal inclusions, and disruptions in cellular organelles. Physicians should be able to understand what each of these findings indicates. However, these are not always specific to a certain disease, and instead most of them are commonly found in many of muscle diseases. Thus, muscle pathological findings should be carefully interpreted under the given clinical settings.

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight and bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded +7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox-positive staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Molecular Mechanisms of Diaphragm Myopathy in Humans With Severe Heart Failure

Rationale: Diaphragm weakness impairs quality of life, exercise capacity, and survival in patients with chronic heart failure (CHF) and reduced left ventricular ejection fraction. However, the underlying cellular mechanisms responsible in humans remain poorly resolved. Objectives: We prospectively evaluated clinical, functional (in vivo/in vitro), histological/ultrastructural, and molecular alterations of the diaphragm from patients with CHF receiving a left ventricular assist device compared with patients without CHF undergoing elective coronary bypass grafting (control) in the observational LIPAMUS-HF (Lipsia Diaphragm And Muscle Heart Failure). Methods and Results: Participants (controls=21, CHF=18) underwent cardiopulmonary exercise and spirometry/respiratory muscle testing alongside diaphragm and cardiac imaging. Diaphragm biopsies were phenotyped for mitochondrial respiration, muscle fiber function, histology/ultrastructure, and protein expression. In vivo respiratory muscle function and diaphragm thickness were reduced in CHF by 38% and 23%. Diaphragm biopsies revealed a fiber-type shift and severe fiber atrophy in CHF alongside elevated proteasome-dependent proteolysis (ie, MuRF1 [muscle-specific RING finger protein 1] expression, ubiquitination, ubiquitin-proteasome activity) and myofibrillar protein oxidation, which corresponded to upregulated Nox (NADPH [nicotinamide adenine dinucleotide phosphate oxidase] oxidase; Nox2/Nox4) signaling. Mitochondria demonstrated severe intrinsic functional and ultrastructural abnormalities in CHF characterized by accumulation of small mitochondria and inhibited autophagy/mitophagy. Single muscle fiber contractile function revealed reduced Ca 2+ sensitivity in CHF and there was evidence of RyR1 (ryanodine receptor 1) dysfunction indicating Ca 2+ leak from the sarcoplasmic reticulum. Mitochondrial and Ca 2+ measures corresponded to upregulated Nox4 isoform NADPH oxidase expression. Molecular markers correlated to whole-body exercise intolerance and diaphragm dysfunction/wasting. Conclusions: Patients with CHF demonstrate an obvious diaphragm myopathy independent of disuse or other confounding factors, such as aging, obesity, or hypertension. Diaphragm weakness in CHF was associated with intracellular abnormalities characterized by fiber atrophy, oxidative stress, mitochondrial dysfunction, impaired Ca 2+ homeostasis, elevated proteasome-dependent proteolysis, but inhibited autophagy/mitophagy, which we speculate offers a novel therapeutic molecular target regulated by a Nox-MuRF1/ubiquitin-proteasome-mitochondria-RyR1/Ca 2+ signaling axis. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02663115.

Effect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS

Background: Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles.Methods: Mice were kept under 6-weeks-CIH (alternating 7% and 21% O2-fractions every 30s, 8h/d, 5d/w) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy) and relevant transcripts (qRT-PCR). Beside wildtype (WT) we included inducible-nitric-oxide-synthase knockout mice (iNOS-/-) to evaluate whether iNOS is protective or risk-mediating. Results: In WT soleus muscle, CIH vs. NOX reduced NMJ size (-37.0%, p<0.001) and length (-25.0%, p<0.05) together with fiber CSA of type IIa fibers (-14%, p<0.05) and increased centronucleated fiber fraction (p<0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p<0.001). Compared to WT, iNOS-/- similarly decreased NMJ area and length with NOX (-55%, p<0.001 and -33%, p<0.05, respectively) or with CIH (-37%, p<0.05 and -29%, p<0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p<0.001) or swollen (>6-fold, p<0.001) mitochondria were observed with iNOS-/- vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS-/- massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) >10-fold. None of these morphological alterations with CIH- or iNOS-/- were detected in gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. Conclusion: CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS-/- at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g. motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.